The prothymosin alpha gene family consists of 7 members, 6 of which appear to be pseudogenes. The seventh gene is clearly functional and gives rise to two 1.3 kb mRNAs. Apparently, alternate splicing events in some tissues include a glutamic acid codon in the processed transcript resulting in a protein with one additional amino acid. The functional gene contains 5 exons, which have been sequenced; the 4 introns occur within the 333 bp coding region. Upon analyzing the 5'-ends of all members of the family, no signal peptide sequences were found. Six genes were very closely related with greater that 90% sequence homology at both the nucleic acid and amino acid levels. As a consequence of an upstream initiator codon and a 7 base pair deletion, the most divergent member of the group encodes a totally different protein. These data rule out the possibility the prothymosin alpha, regardless of which gene is expressed, is secreted. Therefore, based on our previous work, prothymosin alpha must be construed as a mature intracellular protein that plays a role in proliferation in a wide variety of cells. Earlier views claiming that prothymosin alpha was a precursor for thymus specific hormone-like peptides called thymosins are incorrect.